Nitrile hydratase and a method for producing amides

Abstract

An objective of the present invention is to provide a nitrile hydratase capable of producing 2-hydroxy-4-methylthiobutyroamide. The present invention provides a novel nitrile hydratase producing alpha-hydroxyamide using, alpha-hydroxnitrile as the substrate, and the encoding DNA thereof. The enzyme can be obtained from Rhodococcus sp. Further, the enzymatic activity of the enzyme can he maintained stably during the reaction. The present invention provides a method for producing amide compounds, the method comprising the step of reacting this enzyme to nitrile compounds. According to the present invention, from hydroxy nitrile compounds, corresponding amide compounds can be produced biochemically without reducing the enzyme activity of nitrile hydratase.

The invention claimed is: 
     
       1. An isolated protein complex consisting of an α-subunit protein and a β-subunit protein, wherein said protein complex is capable of converting:
 a nitrile group of a nitrile compound to an amide group; and 
 wherein the α subunit protein is a protein selected from the group consisting of: 
 (A) a protein comprising the amino acid sequence of SEQ ID NO: 2; 
 (B) a protein comprising the amino acid sequence of SEQ ID NO: 2 in which one to ten amino acids are substituted, deleted, inserted and/or added; 
 (C) a protein having 95% or higher identity to the amino acid sequence of SEQ ID NO: 2; 
 (D) a protein encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1; and 
 (E) a protein encoded by a polynucleotide that hybridizes to the nucleotide sequence of SEQ ID NO:1 under stringent conditions of 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; 
 and wherein the β subunit protein is a protein selected from the group consisting of: 
 (a) a protein comprising the amino acid sequence of SEQ ID NO: 4; 
 (b) a protein comprising the amino acid sequence of SEQ ID NO: 4 in which one to ten amino acids are substituted, deleted, inserted and/or added; 
 (c) a protein having 95% or higher identity to the amino acid sequence of SEQ ID NO: 4; 
 (d) a protein encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3; and 
 (e) a protein encoded by a polynucleotide that hybridizes to the nucleotide sequence of SEQ ID NO:3 under stringent conditions of 6×SSC at about 45° C., followed by one or washes in 0.2×SSC, 0.1% SDS at 65° C. 
 
     
     
       2. The protein complex according to  claim 1 , wherein the α-subunit protein comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
       3. The protein complex according to  claim 1 , wherein the β-subunit protein comprises the amino acid sequence of SEQ ID NO: 4. 
     
     
       4. An isolated protein selected from the group consisting of:
 (A) an α-subunit protein comprising the amino acid sequence of SEQ ID NO: 2; 
 (B) an α-subunit protein comprising the amino acid sequence of SEQ ID NO: 2 in which one to ten amino acids are substituted, deleted, inserted and/or added, wherein said α-subunit protein is capable of forming a protein complex with any β-subunit protein defined below in subsections (F) to (J) and said protein complex is capable of converting a nitrile group of a nitrile compound to an amide group; 
 (C) an α-subunit protein having 95% or higher identity to the amino acid sequence of SEQ ID NO: 2, wherein said α-subunit protein is capable of forming a protein complex with any β-subunit protein defined below in subsections (F) to (J) and said protein complex is capable of converting a nitrile group of a nitrile compound to an amide group; 
 (D) an α-subunit protein encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1; 
 (E) an α-subunit protein encoded by a polynucleotide that hybridizes to the nucleotide sequence of SEQ ID NO:1 under stringent conditions of 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C., wherein said α-subunit protein is capable of forming a protein complex with any β-subunit protein defined below in subsections (F) to (J) and said protein complex is capable of converting a nitrile group of a nitrile compound to an amide group; 
 (F) a β-subunit protein comprising the amino acid sequence of SEQ ID NO: 4; 
 (G) a β-subunit protein comprising the amino acid sequence of SEQ ID NO: 4 in which one to ten amino acids are substituted, deleted, inserted and/or added, wherein said β-subunit protein is capable of forming a protein complex with any α-subunit protein defined above in subsections (A) to (E) and said protein complex is capable of converting a nitrile group of a nitrile compound to an amide group; 
 (H) a β-subunit protein having 95% or higher identity to the amino acid sequence of SEQ ID NO: 4, wherein said β-subunit is capable of forming a protein complex with any α-subunit protein defined above in subsections (A) to (E) and said protein complex is capable of converting a nitrile group of a nitrile compound to an amide group; 
 (I) a β-subunit protein encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3; and 
 (J) a β-subunit protein encoded by a polynucleotide that hybridizes to the nucleotide sequence of SEQ ID NO:3 under stringent conditions of 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C., wherein said β-subunit protein is capable of forming a protein complex with any α-subunit protein defined above in subsections (A) to (E) and said protein complex is capable of converting a nitrile group of a nitrile compound to an amide group. 
 
     
     
       5. The protein complex according to  claim 1 , wherein the α-subunit protein comprises the amino acid sequence of SEQ ID NO:2 and the β-subunit protein comprises the amino acid sequence of SEQ ID NO:4. 
     
     
       6. The protein complex according to  claim 1 , wherein the protein complex is derived from  Rhodococcus sp . Cr4 strain that has been deposited under the accession numbed FERM BP-6596. 
     
     
       7. The protein complex according to  claim 1 , wherein the protein complex is capable of converting 2-hydroxy-4-methylthiobutyronitrile to 2-hydroxy-4-methylthiobutyroamide.